Perturbation of the smooth muscle cells (SMC) in the vascular wall can result in their proliferation and migration that, along with fibrous/fibrofatty accumulation contribute to intimal thickening, and result in atherosclerosis, the major cause of vascular disease. In order to understand the biology of the SMC and the physiological basis for the differences in phenotype of the medial SMCs, we propose to investigate the molecular biology and mechanics of single SMCs from rabbit smooth muscle tissue under normal and experimentally induced pathological conditions. As a prelude to understanding the cellular biology of medial SMCs that form the intimal thickening in atherosclerosis, we propose to determine: a) how variable expression of the myosin isoforms can alter the mechanical function (unloaded shortening, extent of shortening and maximal force production) of SMCs, and b) possible further modification of these properties via the second messenger pathways which are used to regulate these cells. Specifically, we propose to study the physical and mechanical properties of isolated single SMCs from the vascular (carotid, femoral, renal and saphenous), and digestive (stomach and intestine) systems. We will use single SMC RT-PCR to determine the myosin heavy (SMA/B and SMl/2) and light chain (MLC17a/b) isoform composition from the same cell on which the mechanical measurements were made in order to correlate these parameters. The specific aims of this grant are to determine how myosin isoform expression, correlates with filament organization, and stability, and thereby alters cell mechanics in normal and pathological conditions. This entails examining: 1) Cells of known myosin isoform composition to determine how isoform expression and initial cell length affects unloaded shortening velocity, extent of shortening and force production. 2) Tissues and isolated cells using immunohistochemistry to determine if there is unique myosin isoform distribution and localization within SMC's. 3) If second messenger systems can further alter the "inherent" unloaded shortening velocity in cells of known myosin isoform composition. 4) If SMC's, which are involved in the migration and proliferation resulting from experimentally induced atherosclerosis have unique myosin isoform expression and mechanical properties. [unreadable] [unreadable]